The substrates for cellular functions including transcription, replication, recombination, and chromosome division are chromatin, not naked DNA. These functions are essential for the development and health of all organisms. In the initial level of chromosome organization, DNA is compacted into a repeating array of nucleosomes. The detailed positioning of nucleosomes along DNA can be essential for either negative or positive gene regulation. All eukaryotes and all histone-containing archaea contain strong signals for nucleosome positioning in certain regions of their genomes. Distinctive local arrangements of nucleosomes are reported at telomeres and centromeres, in the vicinity of known gene regulatory regions, and at other chromosomal locations; these particular arrangements are believed to be essential for chromosome function. The sequence of the DNA itself strongly biases the positioning of the nucleosomes into which the DNA is wrapped, and certain DNA sequence rules or motifs involved in nucleosome positioning have been elucidated. These findings imply that genomic DNA sequences are evolved to facilitate their function through effects on their chromatin structure. The long term aim of this work is to elucidate the relationship between the molecular architecture of chromosomes and their function. The goal of the present project is to explore the relationship between genomic DNA sequence, nucleosome positioning, and chromosome function. We will address these goals in three ways. In Aim 1 we characterize the role of the strongest nucleosome positioning sequences in yeast, which we have already isolated. In Aim 2 we analyze the role of nucleosome positioning at specific yeast promoters. In Aim 3 we elucidate the signals for translational nucleosome positioning. We create the strongest possible nucleosome positioning sequence, characterize its consequences in vivo, and analyze its mechanics in vitro.